A member of the Rare Diseases Clinical Research Network (RDCRN), the North American Mitochondrial Disease Consortium (NAMDC) has established a network of 17 clinical centers with a mission to improve the diagnosis and care, establish the natural history, support translational research, and investigate treatment of mitochondrial diseases. Mitochondrial diseases are clinically and genetically heterogeneous disorders due to primary mutations in mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). A consortium, acting in close collaboration with the key patient advocacy groups, including the United Mitochondrial Disease Foundation (UMDF), provides an optimal approach to these complex diseases. With support of an NIH American Recovery and Reinvestment Act (ARRA) grant and 7 years of a U54 award, NAMDC has already generated produced a substantial research infrastructure: a powerful Clinical Registry, a Biorepository, a website for education and recruitment of patients, and mitochondrial disease Research Diagnostic Criteria, which provide an essential foundation for clinical projects and trials. From this firm base, 19 productive patient-oriented projects have already sprouted, including: 4 natural history studies; 6 survey studies; and 9 pilot studies. The NAMDC training program has trained 5 clinician-investigators all of whom remain remarkably active in mitochondrial disease research. This competitive renewal application proposes 5 major Research Projects and a new Career Enhancement program which will expand and reap further rewards from the labor invested to date in NAMDC. The Research Projects are: 1) An expansion of the NAMDC Clinical Registry/Longitudinal Study and Biorepository with detailed analyses of at least 6 mitochondrial disorders and novel registry-based natural history studies of at least 3 diseases; 2) Application of next-generation sequencing to identify causative mutations in the ~40% of NAMDC Registry subjects whose diseases are genetically undefined in collaboration with the Mitochondrial disease SEQuence Data Resource (MSeqDR); 3) Development and utilization of new mitochondrial functional assays in tissues collected via minimally invasive techniques; 4) Extension of an advanced genetics study of pyruvate dehydrogenase deficiency with addition of an innovative biomarker newborn screening program and 5) A longitudinal expanded access study of deoxynucleoside therapy for thymidine kinase 2 (TK2) deficiency. We propose to transform our NAMDC Fellowship into a Career Enhancement program; to initiate new pilot studies; and to transition NAMDC NIH financial support to a broader array of funding sources.